Abstract: Recent intensive studies revealed that recurrent somatic mutation is also a key feature of prostate cancer. Notably, mutations in SPOP (Speckle-type POZ protein), which functions as a substrate-interacting adaptor for the Cullin 3-based E3 ubiquitin ligase, occur in 10%-15% of primary human prostate cancers, representing as the molecular feature of one of the seven sub-types of prostate cancer (PrCa). However, the molecular mechanism and physiological role of SPOP in regulating prostate tumorigenesis remains largely elusive. Moreover, although several downstream ubiquitin substrates have been identified in recent years for Cullin 3SPOP, the upstream signaling pathway to control SPOP stability is largely unknown. Hence, the major goal of this proposal is to explore the upstream regulator as well as to uncover a novel tumor suppressor role of the Cullin 3SPOP ubiquitin E3 ligase in controlling tumorigenesis in the prostate cancer setting. To this end, I have obtained preliminary data showing that the deubiquitinating enzyme OTUD3, but not other OTUD family member, specifically interacts and dequbiquitinates SPOP. More importantly, I identified Nanog, a transcription factor that plays a pivotal role in the maintance of self-renewal and stemness of cancerous cells, as a novel ubiquitin substrate of SPOP. This critical finding, for the first time, links the tumor suppressive role of SPOP E3 ligase to the regulation of prostate cancer stem cells. In this proposal, I plan to: 1) Characterize OTUD3 as an upstream regulator that positively regulates SPOP stability largely through deubiquitination of SPOP; 2) Determine the physiological role of Cullin 3SPOP in suppressing prostate tumorigenesis largely through poly- ubiquitination and degradation of Nanog; 3) Determine whether and how SPOP mutation contributes prostate cancer development and metastasis in vivo. The long-term goals of my career are to apply the insights of molecular and cellular biology to understand the physiological significance of deregulated proteolytic pathways that are important in the development of human malignancies, especially in prostate cancer, and to search for proper druggable targets. This K99/R00 award will provide protected time for me to pursue the novel hypotheses of this proposal, obtain new skill sets to execute experiments and solve problems. In addition, the award will also allow me to focus my efforts on independently conducting basic and translational research, and to train future young scientists in the cancer biology field. Should I receive this award, I will pursue this research at Beth Israel Deaconess Medical Center (BIDMC), Harvard Medical School, where authorities in the fields of ubiquitin E3 ligase and prostate cancer research locate. The outcome of the proposed studies will help elucidate whether the newly identified SPOP mutations could directly regulate prostate tumorigenesis in vivo, and whether blocking its downstream pluripotency maintaining transcription factor Nanog could retard prostate cancer progression.